fig01: Generation and phenotype of p63+/L514F miceGene targeting strategy used to generate the p63+/L514F knock-in mice. The L514F mutation is indicated with *. Dotted black lines indicate the sizes of the fragments generated from each allele upon EcoRI (E) digestion. Oligonucleotide primers (arrows) used for PCR analysis and the relative PCR product length, are indicated.Southern blot analysis of ES cell clones using the EcoRI digestion of genomic DNA and the 5′ probe upstream of the recombinant targeted site.DNA sequencing analysis of the L514F point mutation in properly targeted ES cells.In vivo removal of the NEO cassette obtained by crossing chimeras with CMV-Cre transgenic mice, as revealed by PCR analysis performed on tail genomic DNA using one forward (F) and two reverse primers (R1 and R2).H&E staining of sagittal sections of +/+ and p63+/L514F newborn heads. Cleft of the secondary palate is indicated by the arrow.H&E staining of dorsal skin of p63+/L514F mice at P0. Mutant epidermis is thinner and with less developed hair follicles.Three-dimensional computer-aided reconstruction of tooth crown morphology of the first lower molar at P0. The cusps are numbered as buccal (B1, 2, 3), lingual (L1, 2, 3) and distal (4). The axis of the tooth coming in contact with adjacent teeth is indicated: mesial (M) and distal (D). Scale bar: 400 µm.Histological analysis of sagittal sections of the first lower molar at P0. EO, enamel organ; DP, dental papilla; AM, ameloblasts; OD, odontoblasts. Scale bar: upper panel 50 µm, lower panel 40 µm.

Mentions:
To characterize the developmental alterations that occur in AEC syndrome, we generated a knock-in mouse model carrying a leucine to phenylalanine substitution in position 514 (L514F) in the p63 protein (Fig 1A–D). L514 is a highly conserved amino acid in the first helix of the SAM domain, which is mutated to either phenylalanine or valine in AEC patients (McGrath et al, 2001; Payne et al, 2005; Supporting Information Fig S1A). A correctly targeted embryonic stem cell line allowed the mutation to be transmitted through germline to produce heterozygous p63+/L514F mice. p63 messenger RNA (mRNA) was expressed at similar levels in p63+/L514F mutant and in wild-type epidermis (Supporting Information Fig S1B), whereas p63 protein was more abundant in mutant than in wild-type epidermis (Supporting Information Fig S1C-D) consistent with the previously reported p63 accumulation in the skin of AEC patients (Browne et al, 2011; Moretti et al, 2010). No aberrant isoforms were detected either at the RNA or the protein levels (Supporting Information Fig S1C and S1E).

fig01: Generation and phenotype of p63+/L514F miceGene targeting strategy used to generate the p63+/L514F knock-in mice. The L514F mutation is indicated with *. Dotted black lines indicate the sizes of the fragments generated from each allele upon EcoRI (E) digestion. Oligonucleotide primers (arrows) used for PCR analysis and the relative PCR product length, are indicated.Southern blot analysis of ES cell clones using the EcoRI digestion of genomic DNA and the 5′ probe upstream of the recombinant targeted site.DNA sequencing analysis of the L514F point mutation in properly targeted ES cells.In vivo removal of the NEO cassette obtained by crossing chimeras with CMV-Cre transgenic mice, as revealed by PCR analysis performed on tail genomic DNA using one forward (F) and two reverse primers (R1 and R2).H&E staining of sagittal sections of +/+ and p63+/L514F newborn heads. Cleft of the secondary palate is indicated by the arrow.H&E staining of dorsal skin of p63+/L514F mice at P0. Mutant epidermis is thinner and with less developed hair follicles.Three-dimensional computer-aided reconstruction of tooth crown morphology of the first lower molar at P0. The cusps are numbered as buccal (B1, 2, 3), lingual (L1, 2, 3) and distal (4). The axis of the tooth coming in contact with adjacent teeth is indicated: mesial (M) and distal (D). Scale bar: 400 µm.Histological analysis of sagittal sections of the first lower molar at P0. EO, enamel organ; DP, dental papilla; AM, ameloblasts; OD, odontoblasts. Scale bar: upper panel 50 µm, lower panel 40 µm.

Mentions:
To characterize the developmental alterations that occur in AEC syndrome, we generated a knock-in mouse model carrying a leucine to phenylalanine substitution in position 514 (L514F) in the p63 protein (Fig 1A–D). L514 is a highly conserved amino acid in the first helix of the SAM domain, which is mutated to either phenylalanine or valine in AEC patients (McGrath et al, 2001; Payne et al, 2005; Supporting Information Fig S1A). A correctly targeted embryonic stem cell line allowed the mutation to be transmitted through germline to produce heterozygous p63+/L514F mice. p63 messenger RNA (mRNA) was expressed at similar levels in p63+/L514F mutant and in wild-type epidermis (Supporting Information Fig S1B), whereas p63 protein was more abundant in mutant than in wild-type epidermis (Supporting Information Fig S1C-D) consistent with the previously reported p63 accumulation in the skin of AEC patients (Browne et al, 2011; Moretti et al, 2010). No aberrant isoforms were detected either at the RNA or the protein levels (Supporting Information Fig S1C and S1E).